The central objective of this research is to define ways in which drugs interfere with cholinergic systems on subcellular, cellular and system levels, using gas chromatography/mass spectrometry in conjunction with both stable and radioactive isotopic labelling to obtain a dynamic assessment of cholinergic processes and the factors controlling them. The dynamics of high affinity choline transport and acetylation, and of acetylcholine storage and release, will be studied in vitro in synaptosomes, guinea pig myenteric plexus, rat brain slices and other model systems, and in vivo in rats. Postsynaptic mechanisms will be studied by ligand binding techniques, and the coupling of cholinergic receptors to cellular responses will be investigated in the same tissues by measuring changes in phosphatidylinositol turnover in response to muscarinic agonists, partial agonists and antagonists. Compounds to be studied include cholinergic and anticholinergic agents and agents such as choline and physostigmine which are used to promote central cholinergic activity; psychotropic agents such as antidepressants and neuroleptics which commonly have anticholinergic side effects; lithium, which increases acetylcholine turnover and also interferes specifically with phosphatidylinositol metabolism; and a series of chemical probes which we and others have developed to produce specific biochemical lesions in cholinergic systems. These include a group of irreversible muscarinic agonists, irreversible inhibitors of high affinity choline transport, a precursor of a cholinergic false transmitter and a potent inhibitor of vesicular acetylcholine transport. We expect these probes to be valuable in analyzing both presynaptic and postsynaptic regulatory mechanisms, in producing experimental models of pathological states such as Alzheimer's disease, and potentially in the development of new and more specific therapeutic agents.